The α-synuclein protein, encoded by SNCA, has a key role in the pathogenesis of Parkinson's disease and other synucleinopathies. Although usually sporadic, Parkinson's disease can result from inherited copy number variants in SNCA and other genes. We have hypothesized a role of somatic SNCA mutations, leading to mosaicism, in sporadic synucleinopathies. The evidence for mosaicism in healthy and diseased brain is increasing rapidly, with somatic copy number gains of APP reported in Alzheimer's brain. Here we demonstrate somatic SNCA copy number gains in synucleinopathies (Parkinson's disease and multiple system atrophy), focusing on substantia nigra. We selected sporadic cases with relatively young onset or short disease duration, and first excluded high level copy number variant mosaicism by DNA analysis using digital PCR for SNCA, and/or customized array comparative genomic hybridization. To detect low level SNCA copy number variant mosaicism, we used fluorescent in situ hybridization with oligonucleotide custom-designed probes for SNCA, validated on brain and fibroblasts with known copy number variants. We determined SNCA copy number in nigral dopaminergic neurons and other cells in frozen nigra sections from 40 cases with Parkinson's disease and five with multiple system atrophy, and 25 controls, in a blinded fashion. Parkinson's disease cases were significantly more likely than controls to have any SNCA gains in dopaminergic neurons (P = 0.0036), and overall (P = 0.0052). The average proportion of dopaminergic neurons with gains in each nigra was significantly higher in Parkinson's disease than controls (0.78% versus 0.45%; P = 0.017). There was a negative correlation between the proportion of dopaminergic neurons with gains and onset age in Parkinson's disease (P = 0.013), but not with disease duration, or age of death in cases or controls. Cases with tremor at onset were less likely to have gains (P = 0.035). All multiple system atrophy cases had gains, and the highest levels in dopaminergic neurons were in two of these cases (2.76%, 2.48%). We performed selective validation with different probes after dye swapping. All three control probes used showed minimal or no gains (≤0.1% in dopaminergic neurons). We also found occasional SNCA gains in frontal neurons of cases with Parkinson's disease, and the putamen of one multiple system atrophy case. We present evidence of somatic SNCA gains in brain, more commonly in nigral dopaminergic neurons of Parkinson's disease than controls, negatively correlated with onset age, and possibly commonest in some multiple system atrophy cases. Somatic SNCA gains may be a risk factor for sporadic synucleinopathies, or a result of the disease process.

Background Mutations in GBA cause Gaucher disease when biallelic and are strong risk factors for Parkinson's disease when heterozygous. GBA analysis is complicated by the nearby pseudogene. We aimed to design and validate a method for sequencing GBA using long reads. Methods We sequenced GBA on the Oxford Nanopore MinION as an 8.9 kb amplicon from 102 individuals, including patients with Parkinson's and Gaucher diseases. We used NanoOK for quality metrics, NGMLR to align data (after comparing with GraphMap), Nanopolish and Sniffles to call variants, and WhatsHap for phasing. Results We detected all known missense mutations in these samples, including the common p.N409S (N370S) and p.L483P (L444P) in multiple samples, and nine rarer ones, as well as a splicing and a truncating mutation, and intronic SNPs. We demonstrated the ability to phase mutations, confirm compound heterozygosity, and assign haplotypes. We also detected two known risk variants in some Parkinson's patients. Rare false positives were easily identified and filtered, with the Nanopolish quality score adjusted for the number of reads a very robust discriminator. In two individuals carrying a recombinant allele, we were able to detect and fully define it in one carrier, where it included a 55‐base pair deletion, but not in another one, suggesting a limitation of the PCR enrichment method. Missense mutations were detected at the correct zygosity, except for the case where the RecNciI one was missed. Conclusion The Oxford Nanopore MinION can detect missense mutations and an exonic deletion in this difficult gene, with the added advantages of phasing and intronic analysis. It can be used as an efficient research tool, but additional work is required to exclude all recombinants.

PurposeMutations in GBA cause Gaucher disease when biallelic, and are strong risk factors for Parkinson's disease when heterozygous. GBA analysis is complicated by the nearby pseudogene. We aimed to design and validate a method for sequencing GBA on the Oxford Nanopore MinION. MethodsWe sequenced an 8.9 kb amplicon from DNA samples of 17 individuals, including patients with Parkinson's and Gaucher disease, on older and current (R9.4) flow cells. These included samples with known mutations, assessed in a blinded fashion on the R9.4 data. We used NanoOK for quality metrics, two different aligners (Graphmap and NGMLR), Nanopolish and Sniffles to call variants, and Whatshap for phasing. ResultsWe detected all known mutations, including the common p.N409S (N370S) and p.L483P (L444P), and three rarer ones, at the correct zygosity, as well as intronic SNPs. In a sample with the complex RecNciI allele, we detected an additional coding mutation, and a 55-base pair deletion. We confirmed compound heterozygosity where relevant. False positives were easily identified. ConclusionThe Oxford Nanopore MinION can detect missense mutations and an exonic deletion in this difficult gene, with the added advantage of phasing and intronic analysis. It can be used as an efficient diagnostic tool.

The eNOS G894T and ACE I/D polymorphisms are associated with an increased risk of developing ACS after adjusting for classical risk factors for atherosclerosis in the Bulgarian cohort.